【作者】 蓝恭斌；

【导师】 杨罗艳；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 膀胱癌是我国泌尿生殖系统最常见的恶性肿瘤,发病率和死亡率均位居泌尿系统肿瘤的首位,严重危害我国人民的生命和健康。临床上对于膀胱癌的治疗策略主要是采用手术切除为主,辅以灌注化疗和免疫治疗的综合治疗,但效果不理想。寻找膀胱癌的发病基因,了解膀胱癌的分子遗传学机制,为膀胱癌的诊疗提供理论基础是目前的研究热点。MicroRNA (miRNA)是在真核生物中发现的一类内源性的具有调控功能的非编码单链小分子RNA,大小约22个核苷酸,通常在转录后水平调控基因表达。大量研究表明,miRNA参与肿瘤细胞的增殖、分化、侵袭、转移及凋亡等各个环节。MiRNA具有高度的保守性、时序性和组织特异性等特点,预示它们可能是在一个新发现层次上的基因表达调控方式。miRNA的表达谱可作为肿瘤诊断和预后的指标,对其机制的深入探讨有望为膀胱癌诊疗提供新的思路和靶点。miR-490-5p是新近发现的一种在肿瘤中异常表达的重要的microRNA。初步研究发现,其在包括膀胱癌在内的多种恶性肿瘤中表达显著下调,我们推测其在膀胱癌中发挥类似抑癌基因的功能。为了探索miR-490-5p与膀胱癌的关系,我们利用实时定量PCR技术检测其在膀胱癌、癌旁非肿瘤组织、膀胱癌细胞及正常膀胱细胞之间的表达,探讨其与患者临床分期及病理分级之间的关系。通过生物信息学预测、筛选miR-490-5p的靶基因,应用Western blot进行靶基因蛋白的验证。上调miR-490-5p的表达水平,研究其对膀胱癌细胞生物学行为的影响。进而探讨miR-490-5p在膀胱癌中可能的分子机制,以期为膀胱癌的早期诊断和治疗提供新的策略。第一章miR-490-5p在膀胱癌组织和细胞中的表达及与临床分期、病理分级的关系研究目的：研究miR-490-5p在膀胱癌组织和细胞中的表达及临床意义。方法：采用实时定量PCR检测20例膀胱癌及其癌旁非肿瘤组织、3株膀胱癌细胞(T24、BIU-87和5637)和膀胱上皮永生化细胞(SV-HUC-1)中miR-490-5p的表达水平,研究其与临床分期、病理分级之间的关系。结果：与配对的癌旁非肿瘤组织相比,癌组织中miR-490-5p的表达在所有样本中均下调。癌组织中miR-490-5p的相对表达量为1.175士0.643,癌旁非肿瘤组织中为8.539±1.920,两者表达有统计学差异(P<0.05)。20例膀胱癌组织标本中,根据临床分期分组,Ⅱ期miR-490-5p的相对表达量为1.149±0.677,Ⅲ-Ⅳ期为1-214±0.632,两者表达无显著差异(P>0.05)。根据病理分级分组,高分化组中miR-490-5p的相对表达量为1.770±0.452,中-低分化组中为0.688±0.206,两者表达有统计学差异(P<0.05)。正常膀胱细胞系SV-HUC-1中miR-490-5p的表达显著高于三种膀胱癌细胞(T24.5637、 BIU87)(P<0.05),且随着膀胱癌细胞恶性程度的增加,miR-490-5p的表达渐次降低(5637>BIU87>T24)。结论：1、膀胱癌组织中miR-490-5p的表达水平显著低于癌旁非肿瘤组织,提示miR-490-5p的表达下调可能与膀胱癌的发病机制有关。2、膀胱癌组织中miR-490-5p的表达水平与患者临床分期无关。3、膀胱癌组织中miR-490-5p的表达水平与病理分级相关,随病理分级的增加而降低。4、miR-490-5p在三种膀胱癌细胞系中表达都显著低于膀胱上皮永生化细胞SV-HUC-1,其表达水平与膀胱癌细胞恶性程度有关,随细胞恶性程度的增高而降低。miR-490-5p在T24细胞中表达最低,据此选择该细胞系进行后续细胞功能实验及机制研究。第二章膀胱癌组织和细胞中miR-490-5p靶基因筛选以及验证目的：通过生物信息学检索,预测miR-490-5p的靶基因,并对这些靶基因进行验证方法：通过miRBase.TargetScan和miRanda三个数据库进行检索,筛选miR-490-5p潜在的靶基因：c-Fos、TET1和UVRAG。从实验一结果中选择miR-490-5p表达最低的6例膀胱癌组织、癌旁非肿瘤组织及4株膀胱细胞,进行Western Blot检测,对这三个靶基因的蛋白表达水平进行验证。结果：同膀胱癌旁非肿瘤组织相比,c-Fos蛋白的含量在膀胱癌组织中显著上调(P<0.05),UVRAG的含量显著下调(P<0.05),而TET1在两组间无显著差异(P>0.05)；同正常膀胱细胞相比,c-Fos蛋白的含量在膀胱癌细胞中显著上调(P<0.05),UVRAG的含量显著下调(P<0.05),而TET1无显著差异(P>0.05)。结论：1、miR-490-5p在膀胱癌中发挥着类似“抑癌基因”的作用,其缺失或表达水平下调会导致膀胱癌细胞生长分化,提示其有可能成为膀胱癌基因治疗的又一靶点。2、c-Fos和UVRAG可能是miR-490-5p的靶基因,其对膀胱癌的影响可能通过这两个靶基因发挥作用。第三章miR-490-5p对人膀胱癌T24细胞增殖、侵袭及凋亡影响的实验研究及机制探讨目的：研究上调miR-490-5p表达对膀胱癌细胞T24生物学行为的影响及机制。方法：1、外源性转染miR-490-5p使T24细胞中miR-490-5p表达升高,同时分三组实验：正常组、阴性对照组(转染无意义的mimics)和转染目的miR-490-5p mimics组。2、MTT实验绘制细胞生长曲线,评价miR-490-5p对T24细胞增殖能力的影响。3、Transwell侵袭实验检测各组细胞侵袭能力,评价miR-490-5p对T24细胞侵袭能力的影响。4、Caspase-3方法检测各组细胞凋亡情况,评价miR-490-5p对T24细胞凋亡的影响。5、Western blot检测c-Fos和UVRAG的表达情况,探讨miR-490-5p对膀胱癌影响的机制。结果：1、实时PCR检测结果显示,转染miR-490-5p mimics组T24中miR-490-5p的表达量显著高于阴性对照组(P<0.05)。2、转染miR-490-5p mimics组T24细胞的细胞存活率在48h和72h较阴性对照组低,差异具有显著性(P<0.05)。3、转染miR-490-5p mimics组细胞侵袭数明显低于正常组和阴性对照组,差异具有显著性(P<0.05)。4、Caspase-3酶活力在各组中的情况：正常组为(14.761±1.303)U/mg,阴性对照组为(15.719±1.613)U/mg,转染miR-490-5p mimics组为(43.615±4.399)U/mg。正常组和阴性对照组无显著差异(P>0.05),而转染miR-490-5p mimics组的Caspase-3酶活力显著高于正常组和阴性对照组(P<0.05)。5、转染miR-490-5p mimics组中的c-Fos蛋白表达水平较正常组和阴性对照组显著下降,而UVRAG蛋白表达水平却显著增加(P<0.05)。结论：1、体外细胞实验中,膀胱癌细胞成功导入外源性高miR-490-5p可显著抑制T24细胞的增殖和侵袭能力,促进T24细胞凋亡。2、miR-490-5p通过转录后水平调控c-Fos和UVRAG的表达,影响膀胱癌细胞的增殖、侵袭和凋亡更多还原

【Abstract】 Bladder cancer, with the highest morbidity and mortality among the urinary system tumors, is one of the most common malignant neoplasms of the urinary system in China, which threatens people’s life and health severely. The clinical treatment strategies for bladder cancer is mainly surgical excision, combined with infusion chemotherapy and immunotherapy treatments, which has unsatisfactory results.Find the incidence of bladder cancer gene to understand the molecular genetics of bladder cancer, and provide a theoretical basis for the diagnosis and treatment of bladder cancer is the research focus of the current.MicroRNA is a class of small noncoding single-stranded RNAs found in eukaryotes, about22nucleotides in length, which usually regulates gene expression at the post-transcriptional level. A large number of studies have shown that microRNA participate in tumor cell proliferation, differentiation, invasion and apoptosis. MiRNA are highly conservative, timing and tissue-specific, which indicate that they may be in a new level of gene expression regulation. MiRNA expression profiles can be indicators as a tumor diagnosis and prognosis, and its mechanism of depth is expected to provide new ideas and targets for bladder cancer diagnosis and treatment.miR-490-5p is a newly discovered abnormal expression microRNA in tumors, which has been found in a low level expression in a variety of malignancies including bladder cancer, so we hypothesized that it function as a tumor-supprissive gene.The miR-490-5p expression in bladder cancer and adjacent tissues, three types of bladder cancer cells and normal bladder epithelial cells will be detected by real-time PCR in our project, to explore its relationship with the clinical stage and histological grade. The study will make use of bioinformatics to predict and screen target genes of miR-490-5p, and validate them by Western blot.And then studymiR-490-5p’s influence on the biological behavior of a bladder cancer cell. We hope to clarify the possible molecular mechanisms of miR-490-5p on bladder cancer, so as to provide new strateges on early diagnosis and treatment of bladder cancer.Part I The expression of miR-490-5p and correlation with clinical stage and pathological grade of bladder cancerObjective:To study the expression of miR-490-5p in bladder cancer and clinical significance.Methods:The expression of miR-490-5p was detected by Real-time PCR in20cases of bladder cancer, adjacent non-tumor tissues, three bladder cancer cell lines (T24, BIU-87and5637) and bladder epithelial immortalized cell (SV-HUC-1).The relationship of miR-490-5p with clinical stage and pathological grade was also studied.Results:The expression of miR-490-5p was down-regulated in20samples compared with paired adjacent non-tumor tissue. The relative expression level of miR-490-5p in bladder cancer was1.175±0.643, which was significantly lower than that in the adjacent non-tumor tissue with8.539±1.920(P<0.05).According to the clinical stage, the relative expression level of miR-490-5p was1.149±0.677in Ⅱ phase, and1.214±0.632in Ⅲ-Ⅳ phase, which had no significant difference(P>0.05).According to the pathological grade, the relative expression level of miR-490-5p in well-differentiated group was1.770±0.452, which was significantly higher than that in low-moderately differentiated group with0.688±0.206(P<0.05).The miR-490-5p expression in normal bladder cell line SV-HUC-1was significantly higher than that in the three bladder cancer cell lines (T24, BIU87and5637)(P<0.05), and it gradually decreased (5637> BIU87> of T24) with increasing degree of malignancy of bladder cancer cells.Conclusion:1. The expression of miR-490-5pin bladder cancer was significantly lower than that in adjacent non-tumor tissue, indicating it may be associated with the pathogenesis of bladder cancer.2. The expression of miR-490-5p has no significant correlation with clinical stage in bladder cancer.3. The expression of miR-490-5p has significant correlation with pathological grade, and it gradually decreases with increasing degree of pathological grade in bladder cancer.4. The expression of miR-490-5p in the three bladder cancer cell lines was significantly lower than that in normal bladder cell line SV-HUC-1, and it gradually decreased with increasing degree of malignancy of bladder cancer cells. The expression of miR-490-5p was lowest in T24, and it was selected for further experiment. Part Ⅱ The target gene screen and verification of miR-490-5p in bladder cancer tissues and cellsObjective:To predict and screen target genes of miR-490-5pby bioinformatics, and validate them by Western blot.Methods:By searching databases of miRBase, TargetScan and miRanda.three miR-490-5p potential target genes were selected:c-Fos, TET1and UVRAG. Western blot was used to detect and validate the protein expression levels of these target genes in six cases withlowest expression ofmiR-490-5p, adjacent non-tumor tissue and four bladder cells.Results:Compared with adjacent non-tumor tissue of bladder cancer, c-Fos protein level in bladder cancer significantly upregulated, UVRAG significantly reduced, while TET1with no significant difference between the two groups (P<0.05). Similarly, c-Fos protein level in bladder cancer was significantly upregulated, UVRAG significantly reduced, while TET1with no significant difference compared with normal bladder cells (P<0.05).Conclusion:1. miR-490-5p in bladder cancer plays a similar role of tumor suppressor gene, and its deletion or low expression will lead to bladder cancer cell growth and differentiation, suggesting that it may become another target for bladder cancer gene therapy.2. c-Fos and UVRAG are the candidate target genes of miR-490-5p. miR-490-5p might have exert its impact on bladder cancer through these two target genes. Part Ⅲ Effects of miR-490-5pon cell proliferation, invasion, and apoptosis of human bladder cancer T24cellObjective:To study the effects of miR-490-5p high expression on bilogical characters of bladder cancer T24cell.Methods:1. miR-490-5p expression was increased in T24cell by exogenous transfection, and the experiment were divided into three groups:the normal group, negative control group (transfected meaningless mimics) and group of transfected miR-490-5p mimics.2. MTT assay was employed to draw cell growth inhibition curvesand evaluate miR-490-5p’s impact on the ability of T24cell proliferation.3.Transwell invasion assay was used to determine the miR-490-5p’ sinfluence on the ability of T24cell invasion.4.Caspase-3was detected in each group to evaluate miR-490-5p’s influence on the apoptosis of T24cell.5. c-Fos and UVRAG were detected by Western blot to study the mechanism of miR-490-5p in bladder cancer.Results:1. Real-time PCR results showed that the expression of miR-490-5p in transfected miR-490-5p mimics group was significantly higher than that in the negative control group (P<0.05).2. The rate of T24cell survivalin transfected miR-490-5p mimics group were significantly lower when compared with the negative control group at48h and72hafter transfection (P<0.05).3. Transwell invasion assayshowed that the number of T24invasion in transfected miR-490-5p mimics group were significantly lower than that in the other groups (P<0.05).4. The caspase-3level in each group were as follows:(14.761±1.303) U/mg in normal control group,(15.719±1.613)U/mg in negative control group (15.719±1.613)U/mg, and (43.615±4.399) U/mg in transfection of miR-490-5p mimics group. There was no significant differences between the normal group and the negative control group (P>0.05). The caspase-3levelin transfection of miR-490-5p mimics group was significantly higher than tha in the normal group and negative control group (P<0.05).5. compared with the normal group and negative control group, the c-Fos protein level in ransfected miR-490-5p mimics group decreased significantly, while UVRAG increased significantly(P<0.05).Conclusion:1. In vitro experiments, the bladder cancer cell successfully imported by exogenous miR-490-5p can significantly inhibit proliferation and invasion, and promote apoptosis of T24cells. 2. miR-490-5p exerted imapact on the proliferation, invasion and apoptosis of bladder cancer cells by regulation of c-Fos and UVRAG at post-transcriptional level.更多还原